Clinically, ischemic stroke is recognized as a sexually dimorphic disease. Reproductive hormones are a major contributor to differences in male and female pathobiology. However, emerging data clearly show that outcome from stroke is shaped by biologic sex in addition to hormone exposure. The major hypothesis of this proposal is that distinct gender-based cell death programs are activated after an ischemic insult. We will investigate and identify molecular cell death pathways that are differentially regulated by gender. The overall goal of this proposal is to identify and evaluate the major pathways of cell death activated in male and female brain after in vivo middle cerebral artery occlusion (MCAO). We hypothesize that male cell death is mediated primarily by the caspase-independent activation of nitric oxide (NO), peroxynitrite formation, oxidant-induced DMA damage, and activation of PARP-1, leading to the release of apoptosis inducing factor (AIF) from mitochondria. In contrast, cell death in females is predominately caspase- dependent and secondary to ischemia-induced release of cytochrome C, activation of caspase-9 and formation of the apoptosome. Selective interference with each of these pathways will lead to different outcomes in males and females. In Aim 1we will examine hormonally controlled wild type (WT) and PARP-/- mice to determine the extent of AIF translocation. We will reduce AIF levels in animals of both genders and assess outcomes after stroke. Aim 2 will evaluate cytochrome C release and caspase formation and determine the effect of caspase inhibition in both genders. Gender differences in X-linked inhibitor of apoptosis (XIAP) will be examined in Aim 3. Finally, in Aim 4 we will examine the production of reactive oxygen species (ROS) and bioenergetic markers in males and females. Relevance: Emerging pre-clinical data suggests that the fundamental pathways leading to cell death differ in males and females. Recently, gender differences in the response to pharmaceutical agents used to both prevent (aspirin) and treat (thrombolytics) stroke have been described. Understanding these differences will allow us to develop novel and more efficacious therapies that will enhance the health of stroke patients of both sexes.